Neisseria gonorrhoeae (Gc) is an obligate human bacterial pathogen and the cause of the sexually transmitted infection gonorrhea. Infection with Gc drives the recruitment of neutrophils (PMNs) and production of a characteristic purulent exudate. Although PMNs produce a diverse array of antimicrobial compounds, gonorrheal exudates contain viable and infectious Gc. This observation indicates that Gc has evolved mechanisms to evade PMN clearance, which facilitate Gc persistence and dissemination within a host and transmission to new individuals. To date, the mechanisms used by Gc to survive in the presence of PMNs are poorly understood. Identifying these mechanisms may highlight targets that can be exploited for new therapies to treat gonorrhea, which are urgently needed given the fact that multidrug-resistant Gc is rampant. Investigating Gc resistance to PMNs has been hampered by the fact that Gc does not naturally infect animals other than humans and by the inability of PMN-derived cell lines to make many antimicrobial proteins (APs). Furthermore, Gc infection of human PMNs in suspension does not accurately reflect the adherence- competent, chemokine-primed state of PMNs in acute gonorrhea. Therefore, I developed an in vitro system for examining Gc survival after exposure to attached, interleukin-8 treated, primary human PMNs. In this system, up to 70% of Gc survives initial PMN exposure, with numbers of viable Gc increasing thereafter. I proposed two nonexclusive hypotheses to explain how Gc survives PMN challenge, and in research supported by a K99/R00 award, I gained evidence in support of both. First, Gc is inherently resistant to PMN APs. Second, Gc actively subverts PMNs from releasing APs. In this application, we will extend our preliminary observations in order to identify and characterize the cellular and molecular mechanisms that support Gc survival after PMN challenge. Aim 1 will investigate the release of active APs from granules into phagosomes or at the PMN surface after infection with Gc, and how modulating AP release affects the survival of Gc inside and attached to PMNs. We will examine infected PMNs from human gonorrheal exudates to directly measure the viability of Gc associated with PMNs in vivo and the relationship between bacterial viability and granule release. In Aim 2, we will combine targeted and genome-wide approaches to identify the bacterial gene products that protect Gc after PMN exposure and define the mechanisms by which protection occurs. These gene products include Gc type IV pili and opacity-associated (Opa) proteins, which affect Gc survival after PMN challenge for unknown reasons, as well as gene products known to defend Gc from purified APs. Taken together, the results from this application will reveal the diverse approaches used by Gc to survive in the presence of antimicrobial PMNs, which ensures the continued persistence of gonorrhea within the human population.